WO2004037276A1

WO2004037276A1 - Method and composition for enhancing vascular function

Info

Publication number: WO2004037276A1
Application number: PCT/AU2003/001403
Authority: WO
Inventors: Suzanne Roe
Original assignee: Tarac Technologies Pty Ltd
Priority date: 2002-10-23
Filing date: 2003-10-23
Publication date: 2004-05-06
Also published as: AU2003273614A1

Abstract

A method of improving vascular function characterized in that it consists of administering to a subject in need thereof grape seed extract.

Description

Method and Composition For Enhancing Vascular Function

FIELD OF THE INVENTION

The present invention relates to a method of enhancing vascular function and to a composition for achieving the same. The composition of the present invention is derived from grape seed extract (GSE).

BACKGROUND OF THE INVENTION

Vascular diseases are a scourge of western society. The treatment of vascular disease forms a significant proportion of western healthcare budgets. More importantly, vascular disease contributes to shortened life expectancy and lack of quality of life for many individuals.

Common symptoms of impaired vascular health include hypertension, lack of elasticity in vessel walls and impaired circulation in peripheral blood vessels. A further, less immediately obvious, feature of impaired vascular health is the impairment of functioning of vascular endothelial cells. Located at the interface between blood and a blood vessel wall the vascular endothelium forms the lining of the blood vessel. The endothelium moderates many blood vessel functions and plays a critical role in the mechanics of blood flow and the regulation of coagulation.

Damage to the endothelium can lead to atherosclerotic plaques, the build-up of fatty materials within the walls of the arteries, and possibly to more serious cardiac conditions including angina and heart attacks.

Strategies based on prevention rather than cure are always favoured and much attention has recently been played to the role that plant polyphenols may play in promoting good health and, in particular, the role that they play in promoting vascular health.

Materials rich in polyphenols, notably Green Tea, which is especially rich in catechins, have been associated with antioxidant effects. Green Tea has, in epidemiological studies, been associated with protection from both heart disease and cancer. The vine grape is a preferred commercial source of polyphenol material because of the relative ease with which the material is available for extraction and the availability of significant volumes of the raw material for processing. Vine products yield polyphenols from the grape skins, grape pulp and from grape seeds. Grape seeds have been identified as having the highest concentrations of available polyphenols. Polyphenols present in GSE may be present as monomers, oligomers and polymeric species. Important polyphenolic monomers are typically flavan-3-ols, for example, (-t-)-catechin, (-)-epicatechin, and the gallic acid esters thereof. Oligomeric and polymeric polyphenolic species include procyanidin compounds.

The present invention is concerned with compositions derived from grape seed extract for the promotion of vascular health and process for their production.

SUMMARY OF THE INVENTION

Therefore, according to a first aspect of the present invention, although this need not be the broadest nor indeed the only aspect of the invention there is provided a method of improving vascular function comprising administering to a subject in need thereof grape seed extract.

An improvement in vascular function can be demonstrated by monitoring the ability of vascular tissue to relax following administration of compositions in accordance with the invention.

Vascular function can also be monitored by the measurement of flow-mediated dilation in blood vessels. In a healthy artery, which also has a healthy endothelium, blood vessels are able to relax if the blood flow increases. The endothelium forms the lining of the blood vessel. The endothelium moderates many blood vessel functions and plays a critical role in the mechanics of blood flow and the regulation of coagulation. The ability of a healthy blood vessel to react to changes in blood flow is called flow mediated dilitation.

Generally, an improvement in the flow mediated dilation of vascular tissues would result in an improvement in overall cardiac health. In one form of the invention, the grape seed extract is provided in the form of a powdered product. In alternative forms of the invention the grape seed extract is added to foodstuff products to form a functional food.

As an extract from a natural product that is a known food source GSE is likely to have high consumer acceptance. Further, it is possible to incorporate the GSE into food products in a manner that results in a final food product having attractive taste and flavour characteristics.

Some observers have also noted that FMD decreases after a high fat meal and is related to the triglyceride (TG). High TG levels after food ingestion are also considered a risk factor for coronary disease. In the case where GSE is administered within a food product it is thus possible that the improvement in FMD resulting from the ingestion of GSE may serve to counteract the negative effects of high fat foods.

Preferably, the grape seed extract is administered at a rate of 0.2-5g/day. More preferably still said grape seed extract contains 30-90%w/w polyphenols measured as gallic acid equivalent.

Polyphenols in grape seed extract may be present as oligomeric and polymeric proanthocyanidins. In a preferred form of the invention 60-90%w/w of the total polyphenol component present is oligomeric and polymeric in form and more preferably has 2-16 monomeric units in each polymeric molecule.

It should also be noted that the polyphenols present in the composition of the invention contain less than 1 %w/w f lavonols.

In accordance with a further aspect of the present invention there is provided a method of preparing a grape seed extract useful for improving vascular function and suitable for administration to a subject in need thereof, said method including the steps of:-

separating grape seeds from grapes in a mechanical process;

subjecting said seeds to an extraction process in a suitable solvent to produce a crude extract; and

subjecting said crude extract to one or more purification steps.

Preferably, the one or more purification steps may further include filtration and evaporation of solvent. DESCRIPTION OF DRAWINGS

In the drawings:

Figure 1. Illustrates the relaxation effect of a composition in accordance with the invention on aortic ring preparations pre-contracted at approximate EC₅₀ with noradrenaline.

Figure 2. Illustrates the relaxation effect of a composition in accordance with the invention on aortic ring preparations pre-contracted at approximate EC₅₀ with 8-iso- Prostaglandin E₂.

Figure 3. Illustrates the relaxation effect of a composition in accordance with the invention on mesentery bed preparations pre-contracted at approximate EC₅₀ with noradrenaline.

Figure 4. Shows a comparison of the relaxation effect of a composition in accordance with the invention on myograph preparations precontracted at approximate EC₅o with noradrenaline.

DESCRIPTION OF THE PREFERRED EMBODIMENT

1. Preparation of GSE Products

The GSE product of the invention may be prepared via a range of acceptable process conditions. The preparation of the compositions of the invention can be divided into two separate major processes

a) the preparation of the GSE; and

b) the preparation of a food product incorporating the GSE

Preparation of the GSE involves the separation of grapes seeds from the remaining grape material and the extraction of the polyphenolic material from the grape seeds themselves followed by subsequent processing steps that render the extract into a form suitable for further processing into food products. Mechanical separation processes are used to remove the seeds from remaining grape material.

The extraction of the polyphenolic compounds of interest from the seed products may be achieved by any of a number of chemical extraction processes. For example, solvent extraction of polyphenolics into a suitable solvent such as ethanol and or water may be employed, followed by subsequent solvent removal by evaporation.

The extracted product may then be subjected to a spray drying or freeze-drying step to yield a powdered product. Alternatively, the product may be supplied in liquid form.

The seed product may be incorporated into food products. Typical food products that might incorporate the GSE formed in accordance with the invention include dairy foods such as yoghurt, milk, ice cream and other dairy products, cereals products including breads, biscuits and breakfast cereals; snack food products; fruit juices and other soft drinks, fruit products and confectionary. In addition to this the composition of the invention may be supplied as a more traditional form of supplement for example as a tablet or capsule or liquid tincture.

2. Composition

Products containing phenolic materials can be characterized by subjecting the materials to a number of standard tests that determine and demonstrate the physiochemical properties of the products.

Ferric Reducing Ability of Plasma This test measures the antioxidant capacity of (FRAP Assay) the sample. Reduction of ferric ions to ferrous ions at low pH conditions occurs, proportionate to the reducing ability of the sample. A coloured complex, ferrous-tripyridyl-triazine, is, formed and absorbance at 593 nm measured against known concentration standards.

Total Phenolics This teat measures the total phenolics in a

Folin-Ciocalteau Assay (FC Assay) products against a known arbitrary standard.

Samples are reported as gallic acid equivalents. The FC reagent is an oxidizing agent containing a heteropoly phosphotungstate-molybdate complex. A redox reaction occurs with the sample and a colourimetric assessment of the sample is made at 740 nm.

HPLC determination of Polymeric Reverse Phase HPLC was used to separate Procyanidins individual components followed by uv detection at 280nm. Quantities of gallic acid, catechin, epicatechin and epicatechin gallate were calculated by comparison with standard solutions.

Highly polymerised proanthocyanidins were unresolved. However, peak area of the polymeric peak relative to the total peak area was measured, thereby giving a qualitative measure of polymeric content.

Sub-unit Composition The proanthocyanidin subunit composition, Phloroglucinol Analysis apparent mean degree of polymerisation (mDP) and conversion yield of the samples was determined by acid catalysis in the presence of phloroglucinol.

The above tests were conducted on a sample product produced in accordance with the present invention. In addition, a further sample of the composition produced by the method was subjected to column chromatography to separate the material into fractions of differing polymeric constitution. Table 1 gives details of analyses of a GSE sample produced in accordance with the invention.

In addition other test demonstrated that polyphenols present in the composition of the invention contain less than 1 %w/w flavonols

FRAP Mean % Mass

Purity % area Value Degree of conversio w/w% Approx % polymeric Mmol/ Polymeris n yield of

GAE peak g ation sample

Grape Seed Gallic Acid 0.4

4.7 46 22 2.7 47 Extract Catechin 3.8 FRAP Mean % Mass

GAE peak 9 ation sample

Epicatechin 3.4 Epicatechin gallate 0.3

Gallic Acid ND Catechin 26

Monomeric 8.1 74 Epicatechin 22 ND 1.2 91 Fraction

Epicatechin gallate 2.3

Gallic Acid ND Catechin 0.2

Oligomeric 7.7 68 Epicatechin 0.4 8 3.5 66 Fraction

Epicatechin gallate 0.4

Gallic Acid ND Catechin 0.2

Polymeric 6 57 Epicatechin 0.2 49 4.6 54 fraction

Epicatechin gallate ND

Table 1 Analysis of Composition

3. Effect on Vascular Function

3.1 Relaxation Studies

Several methods of testing the suitability of materials for use in improving vascular health are used. Test conducted on isolated tissue are a rapid method of determining thew usefulness or otherwise of preparations and their effects on vascular tissue. In vitro examination of various vascular tissues of the normotensive WKY rat enable an assessment of the vascular relaxation actions of the compositions of the invention. Tissues examined include isolated aortic ring preparations and preparations of the mesenteric vascular bed. The effects of the compositions of the invention on micro- vessels (100-300 micron diameter) were determined by myograph techniques.

Tests were conducted using the composition of the invention together with the monomeric, oligomeric and polymeric fractions identified above.

Methodology

Twenty week old WKY rats were acclimatised for at least one week and fed normal laboratory chow and water ad libitum. Rats were fasted overnight and euthanased by Nembutal anaesthesia and exsanguination. Aortic tissue and the mesenteric vascular bed attached to the small intestine were carefully excised and placed in Krebs-

Henseleit buffer (pH 7.4) at room temperature gassed with carbogen (95% CO₂:5% O₂) and tissue samples prepared for in vitro experimentation as described below.

The polymeric fraction was to a small degree insoluble in the inert physiological solvents used. Thus, for experimentation the compounds were made up in saline and placed on an orbital mixer for a minimum of 3 h and the solution was centrifuged for 10 min at 3000 rpm. The supernatant was used as the test solution from which serial dilutions were made.

Aortic ring preparation:

Isolated segments of thoracic aorta (3 mm) were mounted under isometric conditions in organ bath chambers and the GSE were screened for ability to cause dose related endothelium-dependant relaxation in vessels precontracted at approximate EC₅₀ values for noradrenaline and 8- so-Prostaglandin E₂as determined previously for WKY rats. The EC₅₀ value is defined as the dose of compound that elicits 50% of the maximally induced effect of a compound - in this case 50% of the total relaxation by the compound after vascular tissue had been precontracted by an agonist.

The experimental protocol involved the following. Rats were sacrificed and the descending thoracic aorta isolated, cleared of adhering tissue and cut into rings, approximately 3 mm in length. The rings were mounted in stainless steel stirrups and place in a 15 ml organ bath chamber. The tissues were bathed in Krebs-Henseleit buffer solution comprised of the following: (mM) 113 NaCI, 4.8 KCI, 1.2 KH₂PO , 1.2 MgSO₄, 25 NaHCO₃, 2.5 CaCI₂, 11.2 glucose and ascorbic acid (0.57) in deionised MilliQ treated water. The buffer solution was continuously bubbled with carbogen and maintained at 37°C. The aortic rings were allowed to equilibrate for 60 min under 4 g of resting tension before contracting with KCI to test the tissue viability. The rings were pre-contracted with approximate EC₅₀ dose of noradrenaline or 8-/so-Prostaglandin E₂. Test GSE were prepared as described above and added cumulatively to the bath. The change in tension was monitored by a computer based data acquisition system (MP100WSW High performance data acquisition unit, BIOPAC Systems Inc.)

The effect of the composition of the invention on a pre-contracted aortic ring preparation are shown in figures 1 and 2. The results clearly demonstrate a dose related contraction I the aortic preparation upon administration of the composition of the invention. Accordingly, the composition of the invention has discernible vasorelaxation properties in large vessels.

Mesenteric Vascular Bed:

The superior mesentery artery was cannulated and flushed with heparin-saline. The entire mesenteric bed, including the intestinal tract was removed and the mesenteric vascular bed was carefully stripped from the gut. The mesenteric preparation was mounted in a 15 mL organ bath and continuously perfused with oxygenated Krebs- Henseleit buffer. After 30 minutes of equilibration, tissue viability was assessed by cumulative intra-luminal injection of various agonists (KCI, noradrenaline). The pressure was raised by the addition of noradrenaline at approximate EC₅₀ in the bath perfusate. Vasorelaxation responses to the composition of the invention as well as the derived fractions of the composition were determined by measuring the extent of pressure reduction. The pressure changes were monitored using MLT844 Physiological Pressure Transducer connected to a pressure amplifier (DA100C, BIOPAC Systems Inc) and a computer based data acquisition system (MP100WSW High performance data acquisition unit, BIOPAC Systems Inc).

The results of the tests are depicted in figure 3. The preparation of the invention demonstrates the ability of the composition of the invention to relax smaller resistance arteries and to mediate the development of higher pressures in smaller arteries. The results are indicative of the ability of the compositions of the invention to lower blood pressure in vivo.

Myograph methodology:

The third arcade of mesenteric arteries of 250-350 micron diameter was cut at the distal end and transferred to the chamber of the myograph and mounted. The vessels were gassed in a closed chamber with carbogen, 37°C for 45 minutes. Normalised vessels were allowed to stabilise for 15 min before testing viability with KCL (40 mM), noradrenaline and acetylcholine. Only vessels that exhibited a contraction greater than 4 mN when exposed to noradrenaline and 25% relaxation to acetylcholine (in the presence of EC₅₀ of noradrenaline) were used for further experimentation.

Myography allows the evaluation of the effect of the compounds of the invention on the contractility properties of micro-vessels. The results of the tests, depicted graphically in figure 4 demonstrate the significant vaso- reactivity of the composition of the invention.

The tests thus demonstrate the ability of the compositions of the invention to induce vaso-relaxation in large blood vessels, in resistance arteries and in micro-vessels. The compositions of the invention therefore clearly have the ability to lower blood pressure and/or increase peripheral circulation of blood in smaller vessels.

3.2 Flow Mediated Dilatation

43 men and women with above average vascular risk from high cholesterol, smoking or high blood pressure were recruited by public advertisement and screened at the

Clinical Research Unit, CSIRO Health Sciences and Nutrition in Adelaide. There were no exclusion criteria on the basis of medication or consumption of alcohol. Subjects were excluded if their BMI was >35 or if they suffered from diabetes mellitus, untreated metabolic disorders such as thyroid or adrenal disease, liver or kidney disease or had unstable coronary artery disease.

The trial was 12 weeks long and consisted of 3 four-weeks periods in a double-blind randomised crossover with control and active ingredients in 240g of yoghurt. Active ingredients consisted of 2g/day of grape seed extract (GSE) in the yoghurt. Blood samples and vascular compliance measures were taken at baseline and at the end of each period. The background diet was a low polyphenol, low quercetin diet. This was achieved by restricting tea and coffee to a maximum of 2 cups per day, restricting apples to one/day and forbidding red wine and onions throughout the 12 weeks. Flow mediated dilatation (FMD) was measured using ultrasound, vascular compliance using radial pulse analysis (HDI).

FMD was assessed in the brachial artery after blockage of blood flow in the forearm with a blood pressure cuff at 200 mm Hg for 5 minutes. The response of the vessel 5 minutes after 100 μg of glyceryl trinitrate sublingually was also assessed. Brachial artery ultrasonography

Brachial artery ultrasonography was carried out in patients after a 12-hour fast and after resting supine for at least 15 minutes in a quiet, temperature controlled room (21 to 25°C). Endothelium-dependent post-ischemic flow-mediated dilation (FMD) and endothelium-independent glyceryl trinitrate (GTN) mediated dilation (GTNMD) were measured During the ultrasound procedure, subjects rested supine in a quiet, temperature-controlled (24°C) room. The left arm was supported comfortably in extension and supination. A high-resolution 12 mHz linear array transducer connected to an Acuson Aspen System (Acuson Pty Ltd., Mountain View, CA, USA) was employed. Continuous EKG monitoring was performed in all studies. The transducer was placed 5 to 10 cm proximal to the antecubital crease and fixed in position by a stereotactic clamp. After good images were obtained, the edge-to-lumen interface was further optimized using depth and gain controls, and an edge enhancement function. Images were recorded continuously on s-VHS videotape (Sony MQSE 180) and 3 second clips were recorded on the ultrasound hard drive for retrospective analysis. A pneumatic tourniquet was placed around the left forearm, and after recording the baseline images for two minutes, the cuff was rapidly inflated to 200 mm Hg for five minutes. Forearm-reactive hyperemia was induced by sudden release of the cuff. Images were recorded continuously from 30 seconds before to 4 minutes after cuff release. A second resting scan was obtained at least 10 minutes after cuff deflation to ensure that the brachial artery diameter returned to the basal level. Two hundred micrograms of glyceryl trinitrate

(GTN) were administered sublingually and the images were recorded continuously for a further five minutes. Maximal FMD and GTNMD responses were calculated as % change in brachial artery diameter from baseline. The analytical (intra-observer) CV of the technique in our hands is in the order of 10%. The CV for repeated within-subject measurement was 15% (N = 10) with a mean ± standard deviation (SD) difference in FMD of 1.6 ± 1.0%.

Statistical analysis

Repeated-measures analysis of variance was calculated with type of yoghurt as the within-subject factor and with sex and order as the between subject factors. Where there was a significant treatment effect detected by repeated measures, paired students f tests were used to locate differences. Bivariate correlation was conducted using Pearson's correlation co-efficient. Analyses were performed with SPSS 10.0 for WINDOWS (SPSS Inc, Chicago). Significance was set at P< 0.05

Results.

12 women and 24 men completed the study and one additional woman missed the last phase of treatment. Six subjects withdrew after commencement and 6 withdrew prior to commencement. All subjects missed 5 days of treatment when the study was temporarily suspended but it was considered that this was of little consequence in a 4 week treatment period.

Flow Mediated Dilatation after compression release and GTN dilatation.

Grape seed extract alone produced an absolute 1.1 % greater dilatation compared with control (p<0.05). GTN induced dilatation was not influenced by GSE.

Baseline GSE Control

Pre compression cm^"2 44.3 ±6.3 45.1 ±6.4 45.5±7.3

Post compression 46.2 ±5.8 47.4 ±6.5 47.3 ±7.3

Change 1.9 ±1.3 2.3 ±1.4 1.8 ±1.3

Pre GTN 44.8±7.1 45.8±7.1 46.2±7.1

Post GTN 52.1 ±6.8 52.7±6.9 52.8±7.0

Change 7.3±2.4 6.9±2.3 6.5±1.8

Table 3 Flow Mediated Dilation as measured by Ultrasound

The results confirm that the GSE composition of the invention and as prepared as described hereinabove favourably influences the endothelium enhancing nitric oxide production, release or slowing down oxidative destruction. The results also demonstrate that sufficient proanthocyanidins from GSE are absorbed to influence flow- mediated dilatation.

A range of other indicators of endothelial and vascular health were also tested and were not adversely affected by treatment with the composition of the invention.

The invention has been described by way of example. The examples are not, however, to be taken as limiting the scope of the invention in any way. Modifications and variations of the invention such as would be apparent to a skilled addressee are deemed to be within the scope of the invention.

Claims

1. A method of improving vascular function characterized in that it consists of administering to a subject in need thereof grape seed extract.

2. A method according to claim 1 , characterized in that the improvement comprises a reduction in blood pressure.

3. A method according to claim 1 , characterized in that the improvement is an improvement in vascular epithelial cell health.

4. A method according to claim 1 , characterized in that the improvement is an improvement in vessel elasticity.

5. A method according to claim 1 , characterized in that the grape seed extract is provided in the form of a powdered product.

6. A method according to claim 1 , characterized in that the grape seed extract is provided in the form of a food additive in a functional food.

7. A method according to claim 1 , characterized in that the grape seed extract is administered at a rate of 0.2-5g/day.

8. A method according to claim 1 , characterized in that the grape seed extract contains 30-90%w/w polyphenols measured as gallic acid equivalent

9. A method according to claim 1 , characterized in that the grape seed extract contains polyphenols present as oligomeric and polymeric proanthocyanidins.

10. A method according to claim 1 , characterized in that the grape seed extract contains 60-90%w/w of the total polyphenol component present as oligomeric and polymeric in form

11. A method according to claim 10, characterized in that the polyphenol component present as polymeric in form having 2-16 monomeric units in each polymeric molecule.

12. A method of preparing a grape seed extract useful for improving vascular function and suitable for administration to a subject in need thereof, said method including the steps of:- -separating grape seeds from grapes in a mechanical process; -subjecting said seeds to an extraction process in a suitable solvent to produce a crude extract; and -subjecting said crude extract to one or more purification steps.

13. A method of preparing a grape seed extract according to claim 12, characterized in that, the one or more purification steps may further include filtration and evaporation of solvent.

14. A method of preparing a grape seed extract according to claim 12, characterized in that the grape seed extract contains 30-90%w/w polyphenols measured as gallic acid equivalent

15. A method of preparing a grape seed extract according to claim 12, characterized in that the grape seed extract contains polyphenols present as oligomeric and polymeric proanthocyanidins.

16. A method of preparing a grape seed extract according to claim 12, characterized in that the grape seed extract contains 60-90%w/w of the total polyphenol component present as oligomeric and polymeric in form

17. A method of preparing a grape seed extract according to claim 12, characterized in that the polyphenol component present as polymeric in form having 2-16 monomeric units in each polymeric molecule.

Dated: 23 October 2003 Tarac Technologies Pty Ltd By their Patent Attorneys LESICAR PERRIN